Unit 3 - Active Recall Flashcards

Question

What is familial Down syndrome? Define Robertsonian translocation.

Answer 1

FAMILIAL DS: - An extra copy of chromosome 21 is attached to another chromosome (e.g. 14 or 15). - Account for 3-4% of cases. - Arise in offspring of parent who carry a chromosome that underwent Robertsonian translocation - Translocation carrier: 45 chromosomes, one of which is a translocation chromosome...Normal phenotype, does not have Down syndrome. DEFINTION - Robertsonian translocation = exchange of long arms of non-homologous acrocentric chromosomes

Answer 2

Yes! plants tolerate it better than animals.. Usually viable; phenotype maybe altered and fertility reduced.

Answer 3

- For diploid (2n) individuals, polyploidy is the presence of more than two sets of chromosomes. i. Triploids - 3n; ii. Tetraploids - 4n; iii. Pentaploids - 5n; and so on…… - Common in plants, less common in animals (some fishes, reptiles, amphibians and invertebrates). Not known in mammals and birds; presumably lethal. - Polyploidy is very important in plants. 30-35% of Angiosperms evolved via some form of polyploidy.

Answer 4

1) Autopolyploid: Multiples of the same genome. e.g., autotetraploid - 4n - can occur during mitosis or meiosis - Nondisjunction of ALL chromosomes during mitosis in early embryo can produce autotetraploid Ex: - Diploid gamete + normal gamete = autotriploid (3n). - Diploid gamete + Diploid gamete = autotetraploid (4n). 2) Allopolyploid : Multiples of closely related genomes e.g., allotetraploid - 4n; 2n from species i and 2n from species ii

Answer 5

- Usually sterile (odd-numbered ploidy). - Most gametes produced are genetically unbalanced.

Answer 6

1. Wheat - cell volume correlated with nucleus volume, correlated with genome size. - Polyploids often have bigger leaves, fruits, seeds. - Bread wheat is a polyploid derived from 3 species. 2. Produce - Production of larger fruits, e.g. strawberries and grapes. - Production of seedless fruit (sterile), e.g. bananas, grapes and watermelon.

Answer 7

- Domestic bananas (mostly 3n = 33) are derived from 2 wild species: Musa acuminata (‘A’) and Musa balbisiana (‘B’). - ‘Gros Michel’, Cavendish are AAA - Most plantains are ABB or AAB - World production = 100 Mt - Most varieties derived from spontaneous hybrid polyploids found in the wild - 2n gametes from one species, 1n gamete from another Overall: - In 1950s & 1960s, Gros Michel wiped out by ‘Panama disease’ (Fusarium)...Replaced by resistant Cavendish. - In 1980s, a new strain of Fusarium, ‘Tropical Race 4’ appeared in Malaysia, now spreading around world. Cavendish has no resistance.

Answer 8

- Rare because DNA replication occurs with high fidelity. - Common because there is a lot of DNA being replicated! (e.g., ~64 new mutations/human generation) - Ultimate source of all genetic variation.

Answer 9

- Somatic mutations are not transmitted from one generation to another. - Germ-line mutations may be transmitted to ~50% of offspring

Answer 10

1. Silent (aka synonymous): no change in amino acid (aa) sequence. Happens in reading frames because of redundancy in genetic code. 2. Missense (aka nonsynonymous): mutation causes 1 aa to be substituted for another, changing the aa sequence. 3. Nonsense: An amino acid codon is converted into a stop codon.

Answer 11

Recall: Indels cause frameshifts that alter reading frames, creating either nonsense or missense effects on protein…. EXCEPT when indels occur as multiples of 3 nucleotides. In such cases the amino acid sequence will change (either become shorter or longer), but the reading frame is preserved. Indels outside of reading frames usually have no effect on phenotype.

Answer 12

1. Loss-of-function: protein function completely or partly lost. Recessive inheritance. 2. Gain-of-function (aka radical): new gene product, or gene product in ‘wrong’ tissue. Dominant inheritance. 3. Neutral: Missense mutation that results in non-significant change in protein function, because one chemically similar amino acid substituted for another, or occurs in a part of the protein that is not important for function

Answer 13

Because of the nature of the chemical changes leading to mutations, transitions are more common than transversions, even though there are twice as many possible transversions.

Answer 14

Forward mutation: alters wild phenotype Reverse mutation changes mutant phenotype back to wild phenotype. i. Intragenic supressor mut. (same gene) ii. intergenic supressor mut. (Dif gene) Note: - Suppressor mutations: first mutation is suppressed by a second mutation

Answer 15

1. Tautomeric shifts (base tautomers) during DNA replication. 2. DNA strand-slippage during DNA replication. 3. Misalignment of homologous chromosomes during crossing-over (recombination) at meiosis I.

Answer 16

1. Ionizing radiation: cosmic rays, X-rays and gamma rays. - change stable molecule into a free radical or an ion, which can alter the structure of bases and break phosphodiester bonds in DNA. 2. Ultraviolet radiation (from sunlight) - Ultraviolet (UV) radiation is electromagnetic radiation of lower energy than ionizing radiation. Can still generate free radicals under some circumstances, but less likely to do so than higher energy radiation. * Can be generated by various types of lamps, e.g., mercury vapour lamps. - PYRIMIDINE DIMERS (TT or CC) THYMINE DIMERS INDUCED BY ULTRAVIOLET RADIATION

Answer 17

Nucleotide excision repair: 1. Protein recognizes mismatches 2. Unwinds DNA in area of mismatch 3. Excises out nucleotides 4. Fills in correct nucleotides

Answer 18

1. Base analogs. - Chemicals that appear similar to the normal bases in DNA, but causes incorrect base-pairing and introduce point mutations during DNA replication. - EX: 5-BROMOURACIL...A nucleotide analog that resembles both thymine and cytosine. Like thymine, 5-bromuracil normally base pairs with adenine, but when ionized it will base pair with guanine. 2. Base modifying agents. - Chemicals that modify groups on the normal bases in DNA that result in incorrect base-pairing and introduce point mutations during DNA replication. 3. Intercalating agents. - Chemicals that distort the normal stacking of bases in DNA resulting in insertion or deletion of a single base-pair during DNA replication. - EX: Intercalating agents insert between adjacent bases distorting them by 0.68 nm, the size of a base. First round of DNA replication, the DNA polymerase randomly selects any nucleoside triphosphate opposite the intercalating agent...Result: frame-shift due to insertion of a base.

Answer 19

Assay for chemical mutagenicity: - A simple method to measure the reversion of a mutant His- Salmonella bacterial strain to His+ Salmonella wild-type strain by potential mutagens. - His- Salmonella cannot grow on minimal medium lacking the essential amino acid, histidine. - His+ Salmonella will grow on minimal medium. - Increased reversions of His- to His+ Salmonella indicate the chemical is a mutagen, and thus, a potential carcinogen. Note: - Inclusion of rat liver enzymes to mimic the chemical modification of potential mutagens in the human body. - Liver enzymes could make the chemical more or less mutagenic.

Answer 20

- Type I restriction endonucleases, discovered in 1960s, recognize specific DNA sequences and then cleave the DNA sequences…somewhere else. - “Restrict” entry of foreign (i.e., viral) DNA into bacterial cells. - Originally thought to be rare, later found to be very common. - not very useful in molecular biology.

Answer 21

- First reported in 1970. - Thousands now known, hundreds commercially available. - ‘Type II’ REs cleave DNA within the recognition site. - This property has made them incredibly useful in molecular biology.

Answer 22

Sequence of nucleotide bases reads the same on the top strand as the sequence of nucleotide bases reads on the bottom strand of the DNA molecule in 5′ - 3’ direction. Ex: 5' - GAATTC - 3' 3' - CTTAAG - 5'

Answer 23

The most common reason is that the ‘host’ (bacterial cell) methylates a base in every copy of the RE site within its own genome. Note: When we get to CRISPR we will encounter an even more clever bacterial host defence system.

Answer 24

- a method for sorting DNA (& RNA) sequence fragments by size - At neutral pH, DNA molecules are negatively charged because of phosphate groups. - In an electrical field, DNA will tend to move toward the positive electrode. - In addition to agarose and water, the gel contains a buffer that provide ions to allow current flow, and to keep the pH slightly above neutral.

Answer 25

Cannot do electrophoresis in a liquid. Need to make a ‘gel’. Most common kind is made from agarose, an uncharged polysaccharide purified from agar of the seaweed, Agar agar.

Answer 26

1. The gel tray. 2. Prepare barriers to retain the agarose. 3. Pour molten agarose into the tray. 4. Insert comb to form the wells before the agarose solidifies. 5. Load DNA samples in individual wells and apply voltage.

Answer 27

- Shorter DNA fragments migrate more rapidly through the gel-matrix than longer molecules. - Migration rate of a linear DNA molecule is inversely related to log of its molecular mass (or # of base-pairs [bp]). - A ‘standard curve’ of known size DNA fragments can be used to extrapolate the size (bp) of an unknown DNA fragment. - Often the 1st stage in the characterization of an unknown DNA molecule. - Based on mobility, can extrapolate the molecular mass (or bp) of an unknown DNA molecule

Answer 28

1. Agarose concentration in gel. - As agarose concentration increases, pore size in gel matrix decreases. - Smaller pores more resistant to DNA movement, favouring small DNA fragments, and giving better resolution of size differences of small fragments. 2. Topology (physical conformation) of DNA molecule. - ie: linear (2nd fastest), relaxed circular (slowest) or supercoiled circular (moves fastest) - In cells, DNA is often negatively supercoiled. - Positively supercoiled DNA can be produced in vitro. 3. Voltage. - Greater voltage speeds up migration rate of DNA fragments during agarose gel-electrophoresis

Answer 29

The %GC or sequence of a DNA molecule For example, two DNA molecules of equal molecular mass (# of base-pairs), one containing 25% GC and the other 75% GC, will migrate through the gel-matrix at the same rate.

Answer 30

IMAGERY: - The image above showed DNA bands in a polyacrylamide gel that were produced after simian virus 40 (SV40) was cleaved by the first known Type II restriction endonuclease. - The DNA bands were radioactively labeled. - This showed how Type II REs could be used to cut DNA sequences in predictable ways. REVOLUTIONIZED: 1. First easy way to study variation in DNA sequences, and map particular features. 2. For first time, could easily recombine DNA sequences (with help of ligases) to create novel DNA sequences. This was the birth of recombinant DNA technology. Note: Addition of agarose gel electrophoresis and EtBr staining also helped a great deal (= easier lab methods)

Answer 31

- A strand of DNA to act as a template - A short, single strand of DNA complementary to part of the template (the ‘primer’) - DNA polymerase - Deoxyribonucleoside triphosphates (dNTPs) - Mg+ (needed by polymerase) Note: - DNA synth. proceeds in 3' direction

Answer 32

HISTORY - Polymerase chain Reaction (PCR) invented by Kary Mullis in 1983, patented in 1985, published in 1986. - led to Nobel Prize and Japan Prize in 1993. - Has been called the most important technique in all of molecular biology. INSIGHT - Mullis’ insight: enzymatic copying of double-stranded DNA using 2 primers, complementary to opposite strands could lead to exponential increase in amount of target sequence.

Answer 33

- Generally, ~30-35 cycles. This allows (in theory*) for more than a billion-fold amplification of target DNA. - Temperature cycling is accomplished using computer-controlled heating/cooling devices (‘thermal cycler’ or ‘PCR machine’)

Answer 34

1. Denaturation - Temperature: 94-96°C - Double stranded DNA denatures (‘melts’) -> single stranded DNA 2. Annealing - Temperature: 50-65°C (dependant on the annealing/melting temperature of primers; Tm) - Primers bind to their complementary sequences - Tm is dependent on length and base composition of primers 3. Extension - Temperature: 72°C - DNA polymerase binds to the annealed primers and extends DNA at the 3’ end of the chain

Answer 35

- Dinucleoside triphosphates, dATP , dCTP , dGTP , dTTP (‘dNTPs’) - Mg2+ (essential for enzyme, affects primer annealing) - Primers (usually, 2) - Template DNA (need not be pure; can be double or single-stranded) - Thermostable DNA polymerase (most often Taq, from Thermus aquaticus)...The ‘original’ version of PCR required addition of DNA polymerase after every denaturation step!.... - Other ingredients: a salt, Tris (pH control), plus stabilizers.

Answer 36

PRIMERS - short molecules of singled-stranded DNA (aka ‘oligonucleotides’ or just ‘oligos’ for short), most often 18-25 b long; can be shorter or longer. PCR PRIMERS - Priming between two oligos annealed to opposite strands can give exponential growth of product. - Size of PCR product depends on how far apart the annealing sites of the 2 primers. - PCR products up to 40 kb have been produced but most PCR involves products 2 kb or less. Yield drops with increasing length of DNA product.

Answer 37

REASON - Primers that are ~18-25b long are long enough to (usually) match only the intended DNA target sequence. OTHER - Successful PCR depends on specific binding of primers to the exact positions that will allow us to amplify our ‘target’ DNA. - Shorter primers are not specific enough in their binding; they may match and bind to multiple positions in the genomic DNA, resulting in amplification of ‘incorrect’ (off-target) DNA sequences. - Longer primers are more costly to make and purchase, but offer little increase in specificity.

Answer 38

1. Amplifying target sequences for further study. - Amplifying a target sequence from within a complex mixture (e.g. genomic DNA sample) is ~equivalent to purifying the sequence of interest. - E.g.: could use to amplify a gene sequence for further study in biomedical, or evolutionary analysis contexts. ---> Must know enough about the sequence of interest to design effective primers 2. Detection of rare DNA sequences. - Can detect as little as a single copy of DNA sequence, even in a complex mixture. RARE APPLICATIONS - bacterial contaminants in food, etc. (e.g., E.coli or Listeria food poisoning) - bacteria in environmental samples - pathogens or endosymbionts in organisms (e.g., HIV, COVID-19) - Forensics – detection of evidentiary DNA at crime scenes - Environmental DNA (eDNA) ---> But NOT good for determining abundance of these rare sequences!

Answer 39

- During early cycles of PCR, production of DNA product is only limited by the amount in the previous cycle --> exponential growth of product. - In later cycles, dNTPs are less abundant, and DNA polymerase may start to wear out, leading to slower growth of product --> ‘linear’ phase. - Eventually, growth in amount of PCR product slows down greatly and then stops, as polymerase and dNTPs start to become exhausted --> ‘plateau’ phase. - Cp value marks first point product exceeds detection threshold of instrument. - When viewed on a log scale, the growth in DNA product during the exponential phase of PCR appears linear. The log-linear phase provides the best information to estimate starting amount of DNA (or RNA) template.

Answer 40

- Growth in amount of PCR product is monitored by using a reporter dye, and a PCR machine capable of detecting fluorescence in each well. - SYBR Green is simplest and cheapest reporter dye. - SYBR fluoresces much more strongly when bound to double-stranded DNA. - Binds primarily to minor groove in double-stranded DNA.

Answer 41

1. Quantify amount of starting DNA of a particular sequence. - E.g. How abundant is a particular type of bacteria (or virus!) in a sample. 2. Measuring rate at which a particular gene is transcribed. - Need to convert mRNA to cDNA first. - Use reverse transcriptase for this.

Answer 42

- Consider what would happen in a DNA extension (synthesis) reaction, in which most of the dGTP is regular dGTP, but a small amount, say 5%, is ddGTP. - In that case, most (95%) of the time when ‘G’ is incorporated, it would be a normal dGTP, and strand elongation past that base could continue. BUT, 5% of the time, a ddGTP would be incorporated, and when that happened, there would be no further extension of that particular DNA strand. - This would give us DNA daughter strands of varying lengths, the lengths of which are determined by where the ‘G’s occur in the sequence. - We could do the same thing for the other bases: ‘Spike’ the DNA polymerization cocktail with small amounts of ddATP, ddCTP, and ddTTP (in addition to the ddGTP). - In this case, we would get a subset of DNA elongation products terminating with a ddNTP base at every position in the DNA sequence.

Answer 43

We attach (different) fluorescent colours to each type of ddNTP (e.g., blue, red, ‘black’, green in this example)

Answer 44

We use gel electrophoresis to sort the fragments by size. The smallest fragments will represent DNA sequences terminating close to the primer.

Answer 45

- Gel electrophoresis uses denaturing polyacrylamide gel (contains urea) to separate single-stranded DNA fragments by size. This type of gel gives very fine resolution, ability to distinguish fragments that differ by 1 base in size. - As ddNTP-terminated fragments migrate in the gel, they pass a laser beam, that excites the fluorescent dyes, and a camera that records the flash of coloured light that results.

Answer 46

PROS - Very accurate - Relatively long sequencing reads (up to nearly 1,000b; although ~650b more common) - Easy to do; can be automated. - Low cost (for small numbers of samples). - Continues to be used for all these reasons. CONS - Too slow for many applications, such as genome sequencing! - Costly when scaled up to acquire lots of data. - Requires purification and preparation of each individual DNA sequence that is being studied. - These limitations led to invention of other methods, so called ‘next-generation’ methods.

Answer 47

- Human Genome Project Consortium in 2000 (total of many labs in 6 countries): 8.64 x 10 7 bases/day (~~10 8 bases/day) - Bentzen lab Illumina MiSeq DNA sequencer: ~1.5 x 10 10 bases/day

Answer 48

The solution: switch to ‘massively parallel’ sequencing Other notes: - Genomes are big! - Handling and sequencing individual samples (DNA templates) is too slow for genome sequencing. - An approach was needed that allow for many (millions) of DNA segments to be sequenced at once ( = ‘massively parallel’)

Answer 49

- DNA needs to be short segments: accomplished by shearing or use of short PCR products. - ‘Adaptor’ sequences are added by ligation to ends of DNA segments. - Adaptors add sites for attachment of DNA sequencing primers and enable attachment to the oligonucleotides on the surface of the flow cell. - DNA segments to sequenced are randomly arrayed across flow cell surface. - ‘Bridge amplification’ used to amplify single DNA molecules into clusters of identical DNA molecules. - Sequencing occurs by addition of fluorescently labeled nucleotide analogs, 1 based at a time. These dNTP analogs are chain terminators (like Sanger) but are reversible (unlike terminators used in Sanger sequencing), so that after chemical treatment of the newly added dNTP, the chain can continue to elongate. - After each dNTP is added, sequencer pauses and exposes flow cell to laser, and takes a picture to record (for each DNA cluster) what base was incorporated in each cluster. Process continues for a few hundred cycles. - Computer interprets the data to infer the DNA sequence within each DNA cluster on the flow cell. - Millions of distinct DNA sequences determined simultaneously this way (= massively parallel DNA sequencing)

Answer 50

Nanopore sequencing - NOT DNA sequencing by synthesis. - Single molecule at a time (no pre-amplification by PCR). - Enzyme unwinds DNA; a single strand is pulled by an electrical current through a pore in a membrane. - Each base produces a characteristic disturbance in electrical current, which can be used to read the base as it travels through the pore

Answer 51

PROS: - Long reads - up to 100kb! - No amplification step to boost amount of template DNA before sequencing. - Small, highly portable DNA sequencer connects to USB port on a computer. - Can be used in the field to get rapid results. - Can detect methylated bases. CONS: - Slightly less accurate than other methods

Answer 52

review slide 35 on DNA sequencing slides 11.3

Answer 53

- Shear genomic DNA into short sequences. - Sequence by next-gen. - Assembler software looks for sequence overlaps between fragments to assemble them into larger fragments (contigs). - Now preferred way of sequencing genomes, but has problems with repetitive DNA sequences. - Long-read sequences (e.g. Nanopore) often used to overcome this problem.

Answer 54

they help with the ‘assembly’ and ‘alignment’ of short reads

Answer 55

- Haploid human genome = ~3.15, Bbp; ~3.2-3.4pg - Roughly equivalent to 7,500 250 page books - A genomic DNA sample includes many thousands copies of whole genome - When a genome is first sequenced, each segment is sequenced, on average, hundreds – thousands of times.

Answer 56

- The number of times a particular base is represented within all the reads from a sequencing run. - Greater read depth gives more confidence a base is accurately read – known as ‘base calling’. - When a genome is sequenced for 1st time, read depth is usually several hundreds – thousands. - When additional copies of a genome are sequenced (called ‘resequencing’) much lower read depths are usually sufficient.

Answer 57

1. Isolate mRNA. 2. Convert to cDNA. 3. Shear cDNA, add adaptors. 4. Sequence by next-gen. 5. Bioinformatics software sorts sequences into different genes (the ‘transcriptome’). 6. The number of times each gene appears in the sequence data is a measure of the degree to which that gene was being expressed in the organism/tissue being studied.

Answer 58

- Many genes/DNA sequences can be used to identify species, but the mitochondrial DNA (mtDNA) COI gene is the most widely used for identifying animal species. - Other genes are used for plants and fungi.

Answer 59

- Early studies (1990s) targeted mtDNA using Sanger sequencing. - Used estimates of how fast mtDNA evolves to date key events. - Showed that all living humans have a common female ancestor who lived in either East or South Africa ~150-200 ka. - All non-African humans have a common female ancestor who lived ~65,000 years ago, about when modern humans dispersed from Africa - Modern humans first dispersed from Africa ~65,000 ya - In Eurasia, encountered 2 other species of human, and interbred with them (next gen). - Next gen. good for mixed up and degraded ancient DNA, also good for detecting genetic traces of ancient hybridization in

Answer 60

- Isolate DNA from an environmental sample; - Amplify microbial sequences using 16s rDNA primers; - Sequence using next gen (e.g. Illumina); - run data through databases to see what species are present and in what relative abundance

Answer 61

- All living (and recently alive) multicellular organisms constantly shed small particles containing DNA. - DNA can be isolated from environmental samples (e.g., filtered water). - Using appropriate primers, informative DNA sequences can be amplified (e.g., mtDNA) and then sequenced. Species that are present can be identified via use of species – DNA databases. - Particular species can also be targeted using taxon-specific primers, followed by qPCR. - Can also detect eDNA in air samples!

Answer 62

see slide 15 on Lecture 13 Sequencing Genotyping Applications

Answer 63

- To determine the genetic basis of inherited diseases or phenotypic traits. - To study the relatedness of individuals or populations, and degree of intermixing of populations (population genetics). - To identify individuals (wildlife ecology). - Parentage analysis or inferring pedigrees. - To identify criminals (forensics).

Answer 64

- In the 1980s, Alec Jeffries invented DNA fingerprinting using minisatellite DNA. - Minisatellites consist of ~10-100 bp sequences that are repeated many times (up to thousands) in tandem arrays - Minisatellite arrays (‘loci’) have extremely high allelic variation, due to frequent mutations involving replication slippage errors and/or unequal crossing-over.

Answer 65

- Jeffries’ discovery had many important applications, including studies of parentage, kinship, and criminal forensics. - However, the method relied on a tedious method called Southern blotting, now no longer much used. - Instead DNA profiling is done using microsatellites*. - Microsatellites are similar to minisatellites, but have shorter sequence repeats (< 10 bp; usually, 2-5 bp). - Like minisatellite arrays, microsatellite arrays show much allelic variation, due to slippage mutations. * Microsatellite arrays can be amplified via PCR. Note: * also known as short tandem repeats (STRs) … and also as… simple sequence repeats (SSRs)

Answer 66

- PCR primers designed for flanking sequences. - Primers are fluorescently labeled. Amplify products of different size. - Separate products by electrophoresis. - Genotypes identified by size of product(s)...E.g. 10 / 12, 28 / 31 repeat genotypes - Co-dominant.....Heterozygotes produce 2 bands, this means both alleles are detected. - Usually use same capillary electrophoresis machines used for dideoxy sequencing. - Multiple microsatellites often amplified at same time, using primers labeled in different fluorescent colours. Called ‘multiplex’ analysis. - 13 ‘standard’ microsatellite loci are used in criminal forensics. These detect enough variability to distinguish all human individuals (except for identical twins). Note: - DNA profiling with microsatellites can be used to track and count wildlife

Answer 67

- PCR-based microsatellite genotyping requires only tiny amounts of DNA: ideal for criminal forensics. - DNA-based methods have helped convict criminals and exonerated many more innocent suspects. - Methods are so sensitive, though, that contamination can be a problem.

Answer 68

- Most microsatellite loci have no effect on health; they are selectively ‘neutral’. - Generally occur outside of exons (e.g., in introns or mostly between genes). - In humans, however, a few microsatellites cause disease. In all cases, these loci involve trinucleotide repeats within genes or other important DNA sequences. - All humans have these microsatellite loci; however, healthy humans have versions (alleles) with a small number of repeats. Humans with genetic disorder have versions with too many repeats. These versions cause production of abnormal proteins. - Examples include Huntington’s Disease, myotyonic dystrophy, fragile X syndrome

Answer 69

- Mutations can either create or destroy restriction endonuclease sites. - Gain or loss of restriction sites (restriction site polymorphisms) can be detected using gel electrophoresis. - Restriction site polymorphisms are most commonly caused by single nucleotide polymorphisms (SNPs). - Examples diagramed in this slide are for haploid DNA

Answer 70

- SNPs caused by single base mutations are the most common genetic variations in genomes. - On average, a SNP occurs every 800-1000 bp in human DNA. - Any 2 randomly chosen humans will have different SNP alleles at several million ‘SNP loci’ - > 600 millions SNPs have been catalogued in human genomes from genome sequencing studies; avg. human genome differs from standard ‘reference’ genome at 4-5M sites. - Usually di-allelic (e.g. an ‘A’ or a ‘G’ at a particular position. - SNPs that are close to each other on a chromosome are usually inherited together (because of limited recombination) forming ‘haplotypes’. - A haplotype is an arbitrarily long stretch of DNA characterized by particular alleles at the SNP positions in that sequence. - Current technologies allow many SNPs to be genotyped simultaneously

Answer 71

- ‘SNP chips’, aka microarrays, are designed to allow many (up to ~1- million) SNPs to be genotyped at once. - Use DNA hybridization-based assay to determine genotypes at known SNPs. - Have become the general method of choice for rapidly screening thousands - ~million SNPs (loci) at once. - Will likely be eventually supplanted by super-cheap genome sequencing.

Answer 72

- Aim is to find genetic links (predictors) to diseases (or traits). - Look for SNPs that have alleles correlated with presence of disease/trait. - Many diseases/traits are influenced by many genes and the environment. - Need to survey many SNPs, and many individuals.

Answer 73

- Some diseases/traits are entirely or mostly determined by a single gene. - Examples: cystic fibrosis, sickle cell anemia…ear wax type! - A single gene polymorphism strongly determines how much fat is in ear wax…and whether it is ‘wet’ or ‘dry’ - Same gene polymorphism influences amount of fatty molecules in sweat. Cool example: - During COVID pandemic, it was discovered that some Neanderthal genetic markers influence resistance to COVID

Answer 74

- bacterial defence against foreign DNA now used by molecular biologists as a genetic engineering tool - CRISPR = Clustered Regularly Spaced Palindromic Repeats - CAS = CRISPR ASsociated proteins

Answer 75

- First discovered in 1987; but function as a defence mechanism against foreign DNA (bacteriophages & plasmids) only discovered in 2005. - Designed to target specific DNA molecules, comparable to adaptive immune systems of vertebrates. - Found in 50% of bacterial species, and 90% of archaea. - CRISPR-CAS systems occur in many different versions, but much interest has focused on CRISPR-Cas9 type

Answer 76

I. Spacer Acquisition (aka ‘adaptation’) II. Expression of crRNAs III. Interference Three components: A. Cas9 enzyme B. crRNA C. tracr RNA

Answer 77

NGG (N = any nucleotide) next to the spacer sequence - Not found in the CRISPR DNA array - Simple and common elsewhere

Answer 78

- Jennifer Doudna and Emanuelle Charpentier - The key innovation: substitution of chimeric gRNA in place of natural crRNA and tracrRNA Notes: - ‘chimeric’ = combination of different things - tracrRNA = trans- activating CRISPR RNA - The ~20 bases at the 5’ end of the gRNA are specific to a target sequence in the genome to be edited. - PAM sequences occur frequently in most genomes

Answer 79

- (s)gRNA designed to target a specific sequence in genome. - sgRNA assembles with Cas9 protein to form effector complex. - Effector complex first finds a PAM, then Cas9 unwinds DNA immediately upstream. If target sequence is present, 20 b 5’ end of sgRNA pairs with it. - Cas9 makes double-stranded cut in genome. - Cellular DNA repair mechanisms engaged, with 2 possibilities: I. Broken ends can be rejoined without any template [nonhomologous end joining, NHEJ]. II. Broken ends can be rejoined using a template [homology directed repair, HDR]

Answer 80

- Most common type of repair to double-strand break in DNA . - No template used. - Instead, nucleotides may be randomly inserted or deleted as the cleaved ends of the chromosome are rejoined. - Often results in insertions and deletions (INDELS). - But if no INDEL mutation occurs, Cas9 keeps cutting the site until a mutation does occur... - Resulting frameshifts lead to non- functional alleles --> gene silencing, ‘knockout’.

Answer 81

- Another way to repair double-strand breaks in DNA. - Uses same repair enzymes as in crossing over or recombination. - Can use a homologous chromosome (sister chromatid) as template. - In CRISPR experiments, can inject ‘donor’ DNA (aka ‘guide DNA’) at same time as Cas9-CRISPR to stimulate HDR

Answer 82

- Relatively cheap and easy. - TARGETING! Can design single-guide RNA to target (almost) any sequence desired. - Relatively specific. - ‘Indels’ created by non-homologous end-joining can create gene knockouts to determine gene function/ phenotype. - Can be introduced to intact, living cells (mRNA for Cas9, or intact Cas9 protein, and sgRNA, mRNA translated by cell). - Can introduce Cas9 with donor DNA to stimulate HDR (homology-directed repair).

Answer 83

1. Off-target effects - cleavage sometimes not specific - A modified Cas9 structure has been created to use longer target sequence, but slower acting. - Can be hard to control whether NHEJ or HDR is used. Germline cells have enhanced homology-directed repair (HDR); adding donor template DNA may help. 2. Mosaicism: not all cells edited, different genomes, so get mosaic effect - Delivery of Cas9 not 100% for all cells; challenge for multicellular organisms. - Various approaches for delivery: transfection, microinjection, electroporation! - Embryo injections at single-cell stage

Answer 84

1. Basic research (create gene knockouts): - Disrupt genes to determine unknown gene functions 2. Editing (“hacking”) genomes to meet human needs/desires: - Reversing mutations that cause genetic disorders. - Donor organs from animals - Improved farm animals (e.g., disease resistance). - Domestication of new plants for agriculture. - ‘De-extinction’ (recreation) of extinct species. - Gene drives to eliminate insect-spread diseases (e.g. Malaria)

Answer 85

- In a study published in 2017, Tait-Burkard and collaborators used CRISPR-Cas9 to clip out a section of CD163 that codes for a single protein domain. The receptor, they observed, seemed unperturbed, while both of the two problematic PRRSV species were still blocked from entry into pig cells in vitro. What’s more, the same team reported this year that gene-edited pigs don’t become infected when exposed to the virus. “It is a complete resistance,” Tait-Burkard says. “It truly is a dead end for the virus.” - In another study...Those enzymes, β-glucanase, xylanase, and phytase, break down matter that pigs don’t otherwise digest; the researchers engineered them to be produced in the modified pigs’ salivary glands. Note: See “Designer Livestock” - The endeavor was a success. Months after the embryos’ implantations, 33 live piglets were born, eight of which survived to sexual maturity. In a paper published in eLife a few months ago, the scientists report that the transgenic pigs indeed produced less nitrogen and phosphorus in their feces, had a faster growth rate, and boosted their feed conversion—the proportion of food that turns into meat.

Answer 86

- Cattle with dark coat are heat stressed in some countries. - Some breeds (e.g. Highland) have naturally light coats. - Introducing light colour gene to dark coated breeds (while preserving desirable traits of dark breeds) would take many generations of selective breeding. - Skin cells from male Holstein Friesian cultured. - PMEL (pigment) gene edited with CRISPR. - Cloning used to produce embryos, which were implanted in cows. - Calves were born with light pigment

Answer 87

- Sickle cell anemia and beta thalassemia are caused by mutations in gene coding for ‘adult’ form of haemoglobin. - Affect 5-7 M people world-wide. - Mutation causes red blood cells to ‘sickle’. This confers resistance to malaria, but also causes anemia, pain and tissue damage. - In new treatment, blood stem cells are removed, cultured, and gene that codes for ‘off switch’ for fetal haemoglobin is knocked out with CRISPR using NHEJ. - Edited stem cells are reintroduced to patient. - Cost may be >>$1M! Note: - Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected female Anopheles mosquitoes. It is preventable and curable. - In 2019, there were an estimated 229 million cases of malaria worldwide. - The estimated number of malaria deaths stood at 409 000 in 2019. - Children aged under 5 years are the most vulnerable group affected by malaria; in 2019, they accounted for 67% (274 000) of all malaria deaths worldwide

Answer 88

- A DNA construct contains gRNA sequence, Cas9, a payload gene, and flanking sequences (H1, H2). - Once introduced as one copy, it copies itself to homologous chromosome via HDR. After sexual reproduction, heterozygous offspring are converted to individuals homozygous for gene drive construct. - In this manner, the payload gene can spread rapidly through the population, because of non-Mendelian inheritance. - Could be used to insert gene for resistance to Malaria, or a gene that reduces fertility of mosquito

Answer 89

- Before PCR, cloning was the only way to copy (‘amplify’) DNA sequences. - To make more DNA with high fidelity for further study or manipulation (living cells copy DNA with much more accuracy than PCR). - To produce substances of scientific or commercial value from genes (e.g., numerous enzymes, synthetic hormones, etc...). - To modify the genomes of plants or animals to introduce new, desired traits.

Answer 90

A plasmid is a stable, self-replicating molecule circular DNA molecule. It contains: - Origin of replication. - Selectable markers to identify cells that have taken up the plasmid. - Unique restriction enzyme cleavage sites.

Answer 91

ENZYME / SEQUENCE / ENDS EcoRI / G*AATTC 5' / OVERHANG HinDIII / A*AGCTT 5' / OVERHANG HpaII / C*CGG 5' / OVERHANG HaeIII / GG*CC / BLUNT HindII / GTY*RAC / BLUNT PstI / CTGCA*G 3' / OVERHANG NotI / GC*GGCCGC 5' / OVERHANG * = cleavage site

Answer 92

- Contains a portion of the lacZ+ gene, with a restriction site ‘linker’ that contains numerous unique restriction enzyme cut sites. - [‘unique’ sites = sites found nowhere else on the plasmid] - Antibiotic resistance gene ‘ampR’ (for ampicillin)

Answer 93

- Cut foreign DNA with a restriction enzyme. - Cut plasmid with same restriction enzyme. - Mix cut foreign DNA and cut plasmid DNA. - Use DNA ligase to seal sugar-phosphate bonds.

Answer 94

- ‘Competent’ bacteria are E. coli made receptive to transformation by chemical or electrical treatment. - They are also ‘lacZ-’, meaning they lack the portion of the lacZ gene that is present in the plasmid. - Ligated plasmids containing DNA inserts are used to transform competent cells. - Transformed bacteria are plated out on agar media containing ampicillin, and X-gal (artificial substrate for β- galactosidase). - Bacteria with no plasmid: do not grow (no antibiotic resistance!). - Bacteria with non-recombinant plasmid: produce B-galactosidase, resulting in blue colonies. - Bacteria with recombinant plasmids: (lacZ gene is disrupted by insert) do not produce B-galactosidase, resulting in white colonies.

Answer 95

- Include operon and regulatory sequences to allow expression of genes in bacteria. - Good for production of many enzymes, especially those that originate from bacteria. E.g., Taq DNA polymerase, commercially available restriction enzymes, etc. - Not good for gene products that require post-translational modification, as occurs with many eukaryotic proteins (eukaryotic cells, such as yeast used to make these gene products).

Answer 96

- In 1989, researchers at Memorial University created 1st transgenic salmon. Combined growth hormone gene (cDNA) from Chinook Salmon with promoter and terminator sequences for antifreeze gene from Ocean Pout. - Chinook Salmon growth hormone (GH) similar to Atlantic Salmon GH (95% amino acid (aa) identity, 98% aa similarity). - Ocean Pout antifreeze promoter shown to be constitutively active in Atlantic Salmon, in contrast to normal GH promoter in salmon, which is only active in response to environmental cues (e.g., day length). - Gene construct injected directly into salmon eggs. Some salmon resulting from this procedure had accelerated growth. - Further breeding showed Ocean Pout promoter-Chinook GH construct was stably integrated into Atlantic Salmon genome, allowing creation of a strain that constitutively expresses Chinook Salmon GH. - The gene construct was found to have rearranged itself inside the Atlantic Salmon genome. Part of the promoter region moved to a downstream position (Figure 1b). The fragmented promoter still worked, but at a slightly lower level than unfragmented promoter. - A company, AquaBounty was formed, and transgenic Atlantic Salmon trademarked as AquAdvantage Salmon.

Answer 97

- Several measures taken to reduce risk of transgenic salmon escaping and interbreeding with wild salmon. - Farmed salmon will be triploids, made by pressure treating eggs. Triploid females are sterile. Males can produce sperm, but this risk controlled by using ‘neomales’: sex-reversed females created by methyl testosterone treatment. All offspring will be females. - Salmon are grown in closed systems on land, instead of net pens in the ocean. This is economically feasible because the fish grow so much faster than normal salmon.

Answer 98

Rhizobium radiobacter, transforms, DNA, genes

Answer 99

- toxicity specific to some insects, not toxic to humans & other animals, biodegradable. - Bt gene has been transferred to many plants using R. radiobacter

Answer 100

1. First, cloned Bt gene into an E. coli plasmid to produce large amounts of the gene sequence for later use. 2. Used restriction enzymes to produce DNA sequences containing varying portions of the Bt genes. These were ligated to a neo gene (confers resistance to kanamycin). 3. Constructs inserted into an expression vector (contained promotor and poly(A) consensus sequences needed for proper gene expression in plant cells) 4. neo+Bt-containing plasmids used to transform R. radiobacter* bacteria. Recombination between expression vector and Ti plasmid occurred inside the bacterial cells. 5. Tobacco plant cells infected with transformed R. radiobacter* bacteria. Whole plants regenerated from plant cells. Recombinant plants screened for Kanamycin resistance. (kanamycin is normally toxic to plants) 6. Plants fed to tobacco hornworms to evaluate toxicity. 7. Most-insect toxic plants had ~2/3 of the Bt gene inserted; full length gene was not effective (perhaps not well expressed by plant). Further breeding demonstrated that Bt gene was stably integrated in plant genome. Similar approach used for other plant species (e.g., cotton, tomatoes, corn...)

Answer 101

Molecular toolbox - Restriction endonucleases(REs) repurposed as DNA scissors. - Ligases allow DNA molecules (from different sources) cut by REs to be recombined to produce novel recombinant DNA, including plasmids & other vectors that can transform other organisms. - PCR is a convenient way to make modest amounts of a particular DNA sequencing, but when large amounts of DNA, or the products of genes (enzymes, proteins) are required, cloning is best. - PCR also routinely used to check success of experiments. - Gel electrophoresis used routinely in combination with RE cleavage or PCR to check the success of cloning/transformation experiments.

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